Higher serum occludin after successful reperfusion Is associated with early neurological deterioration

Abstract Aims Early neurological deterioration (END) is an important factor that affects prognosis in patients with acute ischemic stroke. We explored the relationship between serum occludin levels after successful reperfusion and END in patients treated with endovascular thrombectomy (EVT). Methods We prospectively enrolled 120 stroke patients who underwent EVT with successful reperfusion. Enzyme‐linked immunosorbent assay was used to detect the serum occludin levels on admission and within 1 h after successful reperfusion. Receiver operating characteristic curves (ROC) and regression analysis were used to compare the relationship between serum occludin and END after thrombectomy. Results Among the 120 patients, 36 (30%) experienced END. The END group had higher serum occludin levels than the non‐END group after successful reperfusion [4.31 (3.71–5.38) vs 6.32 (5.88–6.99), p < 0.001]. The ROC curve showed that postoperative serum occludin levels had a significant prediction value for END (AUC: 0.86, p < 0.001). Regression analysis showed that serum occludin was an independent risk factor for END in EVT patients (adjusted odds ratio: 4.46, 95% confidence interval: 1.92–10.32; p < 0.001). Conclusions The higher serum occludin levels were strongly related to END after successful reperfusion. Serum occludin may be an independent risk factor for END in EVT patients.


| INTRODUC TI ON
Stroke is a hot topic: It is the number one disease threatening human health and is associated with high morbidity, disability, and strong recurrence. Moreover, acute ischemic stroke (AIS) is the most common type of stroke. 1,2 In the light of scientific advancements, endovascular thrombectomy (EVT) is recommended as a clinical guideline to treat AIS with large vessel occlusion. 3 However, some patients undergoing EVT have a poor prognosis, and the early neurological deterioration (END) is an important factor that affects the 90-day functional outcome. 4 Therefore, understanding the potential mechanism of END may aid preventative decisions. The underlying mechanisms may include symptomatic intracranial hemorrhage (sICH), postoperative re-occlusion, enlarged infarcts, thromboembolic events, and undetermined causes. [5][6][7] Current clinical evaluations are mainly based on neurological function scores and brain imaging examinations, which also present key challenges. First, the accuracy of neurological function scores is poor. Second, imaging examinations require a long time and may not be recognized early on, despite the emergence of the disease. Therefore, there is an urgent need to develop novel detection methods. Although serum markers are widely studied, there is currently a lack of specific markers to this end. 8 Occludin is a structural component of the tight junction protein of the blood-brain barrier (BBB) and plays an important role in maintaining its integrity. 9 Current studies have found that serum occludin is an important marker of BBB destruction in ischemic stroke and is closely related to ischemic stroke. 10,11 Our previous study found that serum occludin levels could be used to identify early hemorrhagic transformation of AIS. 12 However, it remains unclear whether serum occludin was correlated with END for AIS patients who experienced EVT with successful reperfusion.
Herein, we aimed to identify the incidence of END in stroke patients with successful EVT and whether postoperative serum occludin levels were correlated with the occurrence of END.

| Study design
This research was carried out as a prospective observational study.
The study was approved by the ethics committee of Xuanwu Hospital, and all patients provided signed informed consent.
For AIS patients undergoing EVT, blood samples will be collected on admission and within 1 h after successful reperfusion. Thereafter, enzyme-linked immunosorbent assay (ELISA) was used to detect the occludin levels in the patients' blood. We then collected the clinical data of the patients, including general baseline data, postoperative cerebral infarction volume at 24-48 h after onset, neurological function score (NIHSS), END within 24-48 h, and 90-day clinical outcomes (Barthel index). Statistical methods were used to investigate the relationship between serum occludin and END, cerebral infarction volume, neurological score, and 90-day clinical outcomes.
In addition, we conducted stratification according to the infarction volume, NIHSS score, END, and Barthel Index to compare the differences between serum occludin levels among different groups.

| Patient selection
We included 120 patients who underwent EVT with success- Exclusion criteria included (1) no postoperative blood samples; (2) inflammatory or infectious diseases, cancer, hemorrhagic disease, and severe renal and liver failure; (3) patients with missing important data; and (4) patients with failed recanalization.

| Collection of clinical indicators
General baseline information included age, gender, past medical history, baseline NIHSS score, ASPECT score, time from onset to recanalization; intravenous thrombolysis (yes or no), the Trial of Org 10172 in acute stroke treatment (TOAST) classification, occluded vessel, and EVT strategies (mechanical thrombectomy combined with stenting, mechanical thrombectomy only). The prognostic evaluation data include infarct volume within 24-48 h of onset, 24-h NIHSS score, incidence of END, and the Barthel index at 90 days.
Successful reperfusion or recanalization was defined as a modified Thrombolysis in Cerebral Infarction (mTICI) score of 2b or 3. 13

| Cerebral infarct volume
The baseline infarct volume was estimated by CT perfusion imaging. Magnetic resonance imaging diffusion-weighted (MRI-DWI) was used to detect cerebral infarct volume within 24-48 h after EVT. We adjusted the window width and layer thickness, traced the infarction area of each layer along the edge with the mouse, added the area of all layers, and multiplied the layer thickness to obtain the cerebral infarction volume. 14 According to the size of the infarct volume, we defined the small (0-30 ml), medium (31-50 ml), and large (>50 ml) core infarct groups. 15

| Neurological function score
The NIHSS score was used to evaluate the degree of neurological deficit, and the NIHSS score was defined as mild to moderate stroke if the NIHSS score is less than 17 points; the score was above 17 as severe stroke. 16 It was divided into two groups. The NIHSS score ranged from 0 to 40 points and was assessed on site by two trained neurologists.

| 90-day prognosis score
The prognosis assessment was based on the 90-day Barthel index as the evaluation standard and was assessed by on-site assessment or telephone follow-up. A Barthel index >80 was defined as a good prognosis, and ≤80 was defined as a poor prognosis. 17

| Early neurological function deterioration (END)
END was defined as the worsening of neurological dysfunction within 24 to 48 h, and the NIHSS score decreased by ≥4 points. 18 The main causes of END included infarct expansion, symptomatic intracranial hemorrhage (sICH), re-occlusion, thromboembolic events, and undetermined causes. 6,19,20 Moreover, sICH was determined according to the ECASS-III diagnostic criteria (intracranial hemorrhage confirmed by CT or MRI scan within 22-36 h with an increase in NIHSS score ≥ 4 points; intracranial hemorrhage is the main cause of neurological deterioration). 21 Conversely, vascular re-occlusion within 24-48 h was assessed by CTA (CT angiography) or MRA (magnetic resonance angiography). Intracranial hemorrhage was evaluated by non-contrast computed tomography. Infarct expansion and thromboembolic events were evaluated by MRI-DWI. If sICH, re-occlusion, infarct expansion, and thromboembolic events were excluded, we defined END as undetermined causes.

| Collection of blood samples and detection of serum occludin
Venous blood (2-ml venous blood was extracted from the patient within 24 h of onset, placed at room temperature for 2 h, centrifuged, and placed in an EP tube and stored at −80°C until serum was detected). Centrifuge setting parameters: 3000 rpm, 145,000 g, 10 min. A commercially available ELISA kit (Occludin: USCN, China) was used to detect the serum occludin levels.

| Statistical analysis
The data are presented as mean ± standard deviation or median (interquartile range, IQR). For continuous variables, normal distribution was assessed using the Kolmogorov-Smirnov test. Student's t-test or ANOVA test was conducted for normal/Gaussian distribution data. If the data did not exhibit a normal distribution, we used a Mann-Whitney test. For categorical variables, we used a chi-square test or Fisher's exact test. A correlation analysis was used to de-

| Patients baseline characteristics
We screened 120 eligible patients from 148 patients receiving EVT, excluding 15 patients with recanalization failure, 8 patients with loss of follow-up, and 5 patients with loss of blood samples. Among these 120 patients, there were 90 men and 30 women, with an average age of 63 years ( Table 1).

| Serum occludin and END
Among the 120 patients, 36 cases developed END (30%), including 13 cases of sICH (11%), 12 cases of re-occlusion (10%), 3 cases of new stroke (2.5%), 5 cases of enlarged infarcts (4.4%), and 3 cases of unknown cause (2.5%). There were no significant differences in age, gender, previous medical history, baseline NIHSS score, ASPECT score, baseline infarct volume, and time from onset to recanalization between the END and non-END groups. In addition, the baseline occludin levels had a rising tendency in END group than non-END group  Table 2). However, we were surprised to find that the serum occludin levels after successful thrombectomy were significantly higher in the END group than the non-END group [6.32 (5.88-6.99) vs 4.31 (3.71-5.38) ng/ml, p < 0.001] ( Table 3 and Figure 1A).
After adjusting for age, gender, NIHSS score, ASPECT score, stenting, site of artery occlusion, and etiology of stroke, the multivariate regression analysis found that serum occludin levels after thrombectomy were an independent risk factor for END in stroke patients who had undergone successful thrombectomy (adjusted OR = 4.46, 95% CI: 1.92-10.32, p < 0.001) ( Table 4).
Moreover, the results showed that serum occludin changes before and after EVT in the END group were also significantly higher than that in the non-END group [

| Serum occludin and infarct volume
The results demonstrated that there was a significant correlation between serum occludin levels and cerebral infarction volume after thrombectomy (r = 0.698, p < 0.001). Serum occludin levels in both the moderate and large infarct volume groups were significantly higher than those in the small infarct volume group  Table 3 and Figure 1B). Moreover, the ROC curve showed that serum occluding levels had a good predictive value in the medium-large infarct core (AUC: 0.851, 95% CI: 0.764-0.938, p < 0.001), the best cut-off value was 4.74 ng/ml, as seen in Figure 2B.
Preoperative quantitative penumbra data were obtained by CT perfusion image. We collected CTP data from 58 of the 120 patients.
However, the results suggested that there was no correlation between serum occludin levels after EVT and the preoperative ischemic penumbra volume (r = −0.08, p = 0.618).

| Serum occludin and NIHSS score
Serum occludin levels after thrombectomy were found to be significantly correlated with the NIHSS score within 24 h of a stroke (r = 0.52, p < 0.001). Moreover, the serum occludin levels were significantly higher in the NIHSS > 17 group than in the NIHSS ≤ 17 group  Table 3 and Figure 1C).

| Serum occludin and 90-day prognosis
We found that 24-h serum occludin levels correlated with Barthel index (r = 0.529, p < 0.001). In addition, the group with Barthel index ≤80 had higher serum occludin levels than the group with Barthel  Table 3 and Figure 1D).  Table S1.

| DISCUSS ION
This is the first study to explore the correlation between serum occludin levels and the occurrence of END after EVT with successful reperfusion. We confirmed that high serum occludin levels were closely related to END for stroke patients with successful EVT. In addition, postoperative serum occludin levels were correlated with the volume of cerebral infarction, 24-h NIHSS score, and 90-day functional outcome for patients who underwent EVT with successful reperfusion. Generally, END occurs 2-3 days after surgery, and it is difficult to identify it as early as possible through clinical evaluation or imaging methods. 4 Previous studies suggested that END was a key factor leading to poor prognosis at 90 days. As serious complications of EVT, hemorrhage transformation, postoperative re-occlusion, and thromboembolic events are common causes of END, with a high incidence, as assessed primarily by imaging and clinical symptoms in early stages. [22][23][24] Interestingly, our study found that testing the serum occludin levels immediately after successful reperfusion could predict the risk of END in the next 24-48 h, which was clinically important. A similar study reported that BBB disruption was associated with increased mortality after EVT. 25 The difference from this study was that BBB disruption was observed by CT imaging scan.

Previous studies indicated that AIS patients with increased
serum occludin levels at baseline had a high risk of hemorrhage transformation, regardless of non-reperfusion therapy or reperfusion therapy. 12 In addition, several reports also showed the correlation between occludin degradation and ischemic reperfusion injury.
Wang's study showed that MiR-30a inhibitor restored the loss of occludin in microvessels of ischemic stroke rats and attenuate BBB breakdown and ischemic infarction, which conduced better outcome. 26 Xu's study also showed that ischemic reperfusion injury sig-  TA B L E 2 Comparison of baseline data of patients the loss of ZO-1 and of occludin at the inter-endothelial interface, ultimately increasing BBB permeability. 28 The above three studies demonstrated that the degradation of occludin protein caused the increase in BBB permeability with results of disease progression and complications. Our study was consistent with the above views.
We agreed that serum occludin levels would increase when occludin was degraded in ischemic tissue, which can predict or correlate significantly with the occurrence of END after successful reperfusion. However, there are also different views. An interesting study found that TJs in the BBB promote edema formation and infarct size in stroke. Winkler et al. believed that Cldn3 and occludin could not only protect tight junctions and kept the BBB intact in stroke, but also promoted edema and infarction, as they described the ambivalent effects of sealing proteins. 29 However, there is currently a lack of other evidence to support the interesting findings.
Previous animal studies have confirmed that elevated serum occludin levels could reflect the destruction of the BBB. 10,11 The interesting finding of this study is that there was no significant difference in BBB damage between the two groups before reperfusion. After successful reperfusion, the BBB damage in the END group was significantly heavier than that in the non-END group. It may be that the reperfusion injury aggravated the BBB damage. In addition, it may be attributed to the damage of vascular endothelial cells caused by repeated thrombus removal, which increases the destruction of pairwise comparisons found that there were statistical differences between the small infarction group and the medium infarction group, and between the small infarction group and the large infarction group (p = 0.002, p < 0.001); However, there was no difference between the medium infarction group and the large infarction group (p = 0.168).

F I G U R E 1 Serum occludin levels in different subgroups. (A)
The serum occludin levels of the END group were significantly higher than those of the non-END group (p < 0.001). (B) The larger the infarct core volume, the higher the serum occludin levels. The difference between the large infarct core group and the smaller infarct core group was statistically significant (p < 0.001), and the difference between the middle infarct core group and the smaller core infarction group was also statistically significant (p = 0.002). (C) The serum occludin levels in the NIHSS score >17 group were significantly higher than those in the NIHSS score ≦ 17 group; the difference was significant (p = 0.001).
(D) The serum occludin levels of the poor prognosis group (Barthel index ≤ 80) were significantly higher than those of the good prognosis group (Barthel index > 80) (p < 0.001) the BBB during EVT, and further damage to the BBB would lead to the production of inflammatory cytokines (MMP-9/MMP-2/IL-6/ IL1β/TNF-α, etc.), which rapidly increase throughout the brain, and activates multiple injury mechanisms (i.e., oxidative stress, calcium overload, and apoptosis). [30][31][32] Consequently, this creates a vicious circle that promotes the ischemic cascade reaction and aggravates brain tissue necrosis, 30 resulting in END. Therefore, we speculate that BBB injury is closely related to the occurrence of END during reperfusion therapy. In other words, BBB damage caused by EVT may be the main mechanism for the occurrence of END. By detecting the postoperative serum occludin levels, we can judge the damage of the BBB, so as to predict the possibility of END in the future.
For the prediction of END after EVT, in addition to judging by serum markers, brain imaging assessment can also be used, such as early observation of cerebral edema by cranial CT, and intracranial contrast extravasation observed by cranial CT to judge BBB injury, which is helpful to predict the occurrence of END. In addition, cerebral hyperperfusion syndrome is a common type of END, which can be identified early by observing cerebral blood flow velocity through transcranial Doppler (TCD). 33 Studies showed that postoperative cerebral blood flow velocity increased by more than 100% compared with that before surgery, suggesting cerebral hyperperfusion syndrome. 34 We can also use postoperative CTP imaging to observe cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) to identify END. 33 However, the detection of serum occludin may reflect the degree of neurological impairment and determine the degree of disease, reflecting its important clinical value as an auxiliary diagnostic tool. In the future, serum occludin combined with NIHSS score may better predict the disease severity and functional outcome of stroke patients after EVT.

| Study limitations
There are some limitations which should be emphasized when explaining the results. Firstly, the relatively small sample size limits the power of our study. Secondly, some other clinical factors may be also correlated with the END, which needs to be further investigated.
Thirdly, we did not include patients with failed recanalization, so it is not clear whether there is a correlation between serum occludin levels and vascular recanalization. Finally, individual patients were replaced by CT due to missing MRI scans, which led to differences in the infarct volume results. In the future, it will be necessary to add an analysis to clarify the correlation between END and serum occludin in the two groups of patients with successful or failed recanalization and also increase the sample size to confirm the reliability of the results.

| CON CLUS IONS
In conclusion, there was a high incidence of END after successful reperfusion, and the high occludin levels were strongly related to END. It may be an independent risk factor for END for stroke patients who have undergone EVT with successful reperfusion.
Postoperative serum occludin levels were also correlated with the volume of cerebral infarction, 24-h NIHSS score, and 90-day functional outcome after successful EVT.

ACK N OWLED G EM ENTS
This work was partially supported by grants from by China Postdoctoral Science Foundation (2021M692252), Beijing Postdoctoral Research Foundation (2021-ZZ-029), and Talent Construction Fund (PXM2020_014226_000004).

CO N FLI C T O F I NTE R E S T
There are no conflicts among the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.